Molecularly imprinted polymer for detection of pentraxin protein and method for preparing the same

ABSTRACT

The present disclosure relates to a molecularly imprinted structure for detection of a pentraxin protein and a method for preparing the same by synthesizing a reactive group-pentraxin protein ligand complex specifically reacting with the pentraxin protein and being polymerizable with a crosslink agent to detect a pentraxin protein by using the complex. The present disclosure also provides a chip for detection of a C-reactive protein and a method for preparing the same, the chip including a molecularly imprinted layer having excellent sensitivity to a C-reactive protein and an improved binding force to a metal substrate by using click chemistry.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of International Application No.PCT/KR2010/005059 filed Jul. 30, 2010, which claims Korean PatentApplication No. 10-2009-0133048 filed on Dec. 29, 2009, the entiredisclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure relates to a molecularly imprinted structure fordetection of a pentraxin protein and a method for preparing the same,more particularly, to a method for preparing a molecularly imprintedpolymer for detection of a pentraxin protein, the method includingpreparing a reactive group-pentraxin protein ligand (L-S-R), in which areactive group (R) and a pentraxin protein ligand (L) are boundtogether, reacting the reactive group-pentraxin protein ligand (L-S-R)with a pentraxin protein, and then, mixing the reacting material with acrosslink agent and an initiator, and performing a photo-polymerizationor thermal polymerization reaction to prepare a polymer to which aligand molecularly imprinted with a pentraxin protein is bound.

The present disclosure also relates to a chip for detection of aC-reactive protein and a method for preparing the same, the chipincluding a molecularly imprinted layer having excellent sensitivity toa C-reactive protein and an improved binding force to a metal substrateby using click chemistry.

BACKGROUND OF THE INVENTION

A pentraxin protein is one of protein groups. The pentraxin protein isinvolved in calcium-dependent ligand binding and has a planeβ-structure. The word “pentraxin” is derived from the Greek words forfive (penta) and berries (ragos). The pentraxin protein is formed withfive monomers arranged in a regular pentagon shape and has a diameter ofabout 9.5 nm and a height of about 3.5 nm. A relatively small pentraxinprotein includes a serum amyloid P component (SAP) and a C-reactiveprotein (CRP). A relatively large pentraxin protein includes PTX3(cytokine regulatory molecule) and other nervous proteins.

The CRP is one of representative acute-phase reactive proteins fortissue damage or inflammation in mammals. The CRP is used as a diagnosisfactor for various inflammatory diseases and cardiac infarction. The SAPis a vertebrate protein, which is a precursor of the amyloid componentP, and observed in all types of amyloid deposits for glomerular basementmembranes and elastic fibers in blood vessels. The SAP is an importantmolecule marker for the Alzheimer disease and utilized for the diseasediagnosis.

With respect to a conventional method for detection of a C-reactiveprotein, there have been reported a method using an antibody (KoreanPatent Application Publication No. 2004-0064054), a method using aphosphocholine ligand (U.S. Pat. No. 0,246,522 and British Patent No.02217335), and other methods. In case of using the antibody, a high costfor preparing an antibody is required. In order to maintain activity ofan antibody, it is necessary to manage whether the activity isdeteriorated, using a special storage method. In case of using thephosphocholine liqand, an intermediate to fix the ligand to a protein,agarose gel, polystyrene, or others is needed.

In order to overcome the problems, many researchers have put forth theirefforts in developing detection methods using a molecularly imprintedpolymer having excellent stability, compared to an antibody.Accordingly, the present disclosure provides a method for preparing amolecularly imprinted polymer to increase an effect of detection of aC-reactive protein, which is a representative example of pentraxinproteins. To the end, the inventors of the present disclosure developeda novel molecularly imprinting technology using a phosphocholine ligand,by synthesizing styrene-phosphocholine ligands including aphosphocholine group designed to be polymerized with styrene that is acrosslink agent, and inducing a polymerization reaction of the crosslinkagent in the state that the styrene-phosphocholine ligands aresimultaneously bound to five phosphocholine ligand binding sites presentin a C-reactive protein.

BRIEF SUMMARY OF THE INVENTION

In view of the foregoing, the present disclosure provides a molecularlyimprinted structure for detection of a pentraxin protein and a methodfor preparing the same by synthesizing a reactive group-pentraxinprotein ligand complex specifically reacting with the pentraxin proteinand being polymerizable with a crosslink agent to detect a pentraxinprotein by using the complex.

The present disclosure also provides a chip for detection of aC-reactive protein and a method for preparing the same, the chipincluding a molecularly imprinted layer having excellent sensitivity toa C-reactive protein and an improved binding force to a metal substrateby using click chemistry.

In accordance with one aspect of an the present disclosure, there isprovided a method for preparing a molecularly imprinted structure fordetection of a pentraxin protein, the method including: binding apentraxin protein and a reactive ligand having a following structuralformula; chemically reacting the complex of the pentraxin protein andthe reactive ligand on a reactive surface of a support; and removing thepentraxin protein from the reacting material by washing the reactingmaterial with an elution buffer solution.L-S-R  [Structural Formula]L: Ligand for binding with the pentraxin proteinR: Reactive groupS: Linker for linking between the ligand for binding with the pentraxinprotein and a reactive group

In an illustrative embodiment, the pentraxin protein is formed with fivemonomers arranged in a regular pentagon shape and has a diameter ofabout 9.5 nm and a height of about 3.5 nm. Since each one ligand isbound to each of the monomers of the pentraxin protein, five reactiveligands in the embodiment are bound to one pentraxin protein (see FIG.1).

The “reactive ligand” refers to a material where a ligand (L) capable ofbeing bound to a pentraxin protein, which is a target molecule, to forma complex, and a reactive group (R) capable of being bound to a reactivesurface of a support through a polymerization or chemical reaction arelinked by a linker (S). The intermediate linker (S) intermediates thebinding between the ligand (L) and the reactive group (R) and makes thebinding to the support flexible. That is, one end of the reactive ligandis formed with a ligand (L) bound to a target material. The other end ofthe reactive ligand is formed with a reactive group (R) capable of beingpolymerized or bound to a metal material.

The “support” is a solid polymer or a metallic material that serves as abody of a molecularly imprinted structure, and may be in form of aplate, film, nano-particle, nano-wire, porous particle, or bead. Ametallic material support may be prepared separately, while a polymersupport may be prepared in situ through a polymerization reaction with areactive ligand having a polymerizable reactive group.

The “reactive surface” is a layer separated from an aqueous solutionlayer in a buffer solution and refers to a surface of an organicmaterial that can be polymerized reaction (e.g., copolymerization) witha reactive group of a reactive ligand or a surface of a metallicmaterial that can be chemically bound to the reactive group. In theembodiment, for the reactive surface of the organic material, a materialin which styrene and DVB are mixed with a ratio of 9:1 is used. Themixture is not mixed with an aqueous solution and forms a layer on thesurface of the aqueous solution. In this case, the reactive surfacerefers to an interface between the aqueous solution and the layer of themixture of styrene/DVB and is copolymerized with a reactive ligand inthe aqueous solution. After the copolymerization is finished, thereactive surface is modified by the reactive ligand (see FIG. 5).

The words “molecularly imprinted structure” used in illustrativeembodiments mean a imprinted material having a ligand or space capableof binding with target molecule by using target molecular as template.Particularly, the “molecularly imprinted polymer (MIP)” refers to apolymer which is synthesized by using a monomer being bound to a propertemplate as a starting material, and then the template is removed, sothat the polymer has a space in the same shape as that of the template.A template can be inserted into the space having the same shape as thetemplate. A molecule having a different stereo-structure from thetemplate cannot be inserted into the space. Accordingly, it is possibleto separate various molecules, which are different from a template, byusing a polymer having a template space. This is the same theory as theFischer's Lock-and-Key Concept that an antibody formed against anantigen selectively interacts only with the antigen, or the ReceptorTheory that an enzyme in a body exhibits activity only to a certainsubstrate. Many researches for the molecularly imprinting technique havebeen performed in the field of a system for sensing or separating acertain material using a molecularly imprinted polymer.

In an illustrative embodiment, the pentraxin protein may be onepentraxin protein selected from the group consisting of a C-reactiveprotein, a serum amyloid P component, and PTX-3 (Pentraxin 3).Preferably, the pentraxin protein may be, but is not limited to, theC-reactive protein. The “C-reactive protein (CRP)” is one of plasmaproteins that significantly increase in inflammatory diseases ordiseases such as necrosis of body tissues, and a representativecomponent of a so-called acute phase reactive protein. In case ofoccurrence of a problem in a body, the CRP increases in a short timefrom about 6 to about 24 hours. In recovery of a lesion, the CRP rapidlydeclines and disappears in a short time within about 24 hours.Accordingly, the CRP exhibits characteristics that cannot be seen in ageneral immune globulin antibody. As such, CRP measurement is useful fordiagnosis of existence of an inflammatory or historrhexis disease andthe severity thereof, disease progress monitoring, and prognosisprediction.

In an illustrative embodiment, if the pentraxin protein is theC-reactive protein, the ligand (L) of the pentraxin protein may be, butis not limited to, phophocholine. If the pentraxin protein may be theserum amyloid P component, the ligand (L) of the pentraxin protein maybe, but is not limited to, proline. If the pentraxin protein is PTX-3,the ligand (L) of the pentraxin protein may be, but is not limited to,C1q (18 amino acids). For example, if the pentraxin protein is aC-reactive protein, two calcium divalent ions are bound to each monomer.A phosphate group of phosphorylcholine is bound to the monomer, havinghigh selectivity. It is possible to prepare a reactive ligand capable ofdetecting each pentraxin protein by using the above-described ligandsaccording to a chemical method well-known in the art.

In an illustrative embodiment, a molecularly imprinted structure fordetection of a pentraxin protein is prepared by using a C-reactiveprotein that is one of the pentraxin proteins. However, it is obvious toone of ordinary skill in the art that for the other pentraxin proteins,a molecularly imprinted structure can be prepared in the same manner asdescribed above.

Hereinafter, the method for preparing the molecularly imprintedstructure using the C-reactive protein according to an embodiment willbe described.

In an illustrative embodiment, a schematic method for polymerizing asupport in situ is illustrated in FIG. 4. Basically, a C-reactiveprotein, which is one of pentraxin proteins, and astyrene-phosphocholine ligand, which is a reactive ligand of theC-reactive protein, are reacted with each other in advance. Thereafter,the reacting material is added to a buffer solution containing acrosslink agent and an initiator to synthesize a molecularly imprintedpolymer through a photo-polymerization reaction. In another illustrativeembodiment, a schematic method for separately preparing the support andthen binding the support to the reactive ligand is illustrated in FIG.6. The C-reactive protein, which is one of pentraxin proteins, and thestyrene-phosphocholine ligand, which is a reactive ligand of theC-reactive protein, are reacted with each other in advance. Thereafter,the reacting material is chemically bound to a separately preparedsupport in a film, single layer, or particle form.

In an illustrative embodiment, the reactive group (R) may be, but is notlimited to, a polymerizable group or a functional group reacting withmetal or a metal oxide.

The polymerizable group may be, but is not limited to, a radicalpolymerization monomer selected from vinyl, styryl, acryloyl,methacryloyl, itaconoyl, sorbyl, and dienoyl. In an example, styrene isused as the polymerizable group.

The functional group reacting with metal or a metal oxide may be a metalreactive group selected from thiol, disulfide, and thioether, or a metaloxide reactive group selected from trichlorosilyl, trimethylsilyl,triisopropylsilyl, and catechol. However, the present disclosure is notlimited thereto.

In an illustrative embodiment, the linker (S) may be, but is not limitedto, alkyl, aryl, arylalkyl, oligoethylene oxide, or a combinationthereof. However, the present disclosure is not limited thereto. Lengthof carbon chains of the linker (S) may be, but is not limited to,properly selected, for example, in a range of C3 to C20, to createflexibility between the ligand (L) and the reactive group (R). In anexample, alkylcarboxylic ester is used as the linker.

In an illustrative embodiment, the reactive surface may be, but is notlimited to, a surface of a film, nano-particles, a nano-wire, or porousparticles formed of an organic or metallic material.

In an illustrative embodiment, the chemical reaction may be, but is notlimited to, a polymerization reaction of a monomer and a crosslink agentwith a reactive group of a reactive ligand, or a chemical bindingreaction of metal or a metal oxide with a reactive group of a reactiveligand.

For example, the polymerization reaction may be, but is not limited to,a thermal polymerization or a photo-polymerization reaction by a radicalpolymerization initiator or a photo-polymerization initiator.

For example, the chemical binding reaction may be, but is not limitedto, a binding reaction between thiol and gold particles or betweentrichlorosilyl and a metal oxide.

In accordance with another aspect of of the present disclosure, there isprovided a method for preparing a molecularly imprinted structure fordetection of a pentraxin protein, wherein the pentraxin protein is aC-reactive protein, and the method includes:

(a) preparing a styrene-phosphocholine ligand, in which styrene andphosphocholine ligand are bound to each other;

(b) reacting the styrene-phosphocholine ligand with a C-reactiveprotein;

(c) adding the reacting material to a buffer solution containing amonomer, a crosslink agent, and an initiator and mixing them;

(d) performing a UV photo-polymerization reaction for the mixture toobtain a polystyrene polymer to which the C-reactive protein is bound;and

(e) removing the C-reactive protein by washing the polystyrene polymerwith an elution buffer solution.

In an illustrative embodiment, the styrene-phophocholine ligand of theabove step (a) is 12-(4-vinylbenzyl)oxycarbonyl dodecyl phosphocholineprepared according to reaction steps illustrated in FIG. 2 (refer toExample 1).

In an illustrative embodiment, the C-reactive protein and thestyrene-phosphocholine ligand of the above step (b) are reacted with amolar ratio of about 1:3 to about 1:7, and preferably a molar ratio ofabout 1:5.

In an illustrative embodiment, in the above step (c), the monomer andthe crosslink agent are styrene/DVB (divinylbenzene), and the initiatoris DBK (dibenzylketone).

In an illustrative embodiment, the monomer, the crosslink agent, and theinitiator may be mixed with a mixing ratio of about 8 to 18:1:1 to 5. Inthe illustrative embodiment, styrene, DVB, and DBK are mixed with amixing ratio of about 9:1:1.

In an illustrative embodiment, the photo-polymerization reaction of theabove step (d) is performed by performing a UV photo-polymerizationreaction at about 35° C. to about 40° C. for about 1 to about 3 hours,and then, performing a UV photo-polymerization reaction at about 80° C.to about 100° C. for about 1 to about 3 hours.

In accordance with another aspect of the present disclosure, there isprovided a reactive ligand capable of binding to a pentraxin protein,the reactive ligand having the following structural formula:L-S-R  [Structural Formula]L: Ligand for binding with the pentraxin proteinR: Reactive groupS: Linker for linking between the ligand for binding with the pentraxinprotein and the reactive group

In accordance with another aspect of the present disclosure, there isprovided a molecularly imprinted structure for detection of a pentraxinprotein, the molecularly imprinted structure including a support and amultiple number of reactive ligands bound to a reactive surface of thesupport, wherein the multiple number of the reactive ligands aremolecularly imprinted to be bound to the pentraxin protein.

In accordance with another aspect of the present disclosure, there isprovided a chip for detection of a C-reactive protein, the chipincluding: a fixing layer formed on a metal substrate and having anazide or an acetylene end group R¹; and a molecularly imprinted layerformed on the fixing layer and including R²—X-phosphocholine (wherein R²is an azide or an acetylene end group, and X is a spacer group having alength of about 0.1 nm to about 5 nm), wherein the phophocholine groupincluded in the molecularly imprinted layer is arranged to correspond toa phosphocholine binding site of each molecule of the C-reactiveprotein, and the fixing layer and the molecularly imprinted layer may bebound to each other through a click chemistry reaction between the endgroup R¹ of the fixing layer and R² included in R²—X-phosphocholine ofthe molecularly imprinted layer.

In an illustrative embodiment, if the end group R¹ of the fixing layeris the azide group, R² of the molecularly imprinted layer may be theacetylene group. If the end group R¹ of the fixing layer is theacetylene group, R² of the molecularly imprinted layer may be the azidegroup. However, the present disclosure is not limited thereto.

A target material desired to be sensed in an illustrative embodiment isthe C-reactive protein. The C-reactive protein is one of proteinsobserved in blood. Since the C-reactive protein increases in case ofexistence of inflammation, it is used as a marker for inflammation. Thephysiological function of the C-reactive protein relates to binding withphosphocholines presented on surfaces of cells (and some types ofbacteria) that are dead or dying to activate a complement system,through a C1Q complex (antigen+antibody). The C-reactive protein raisesphagocytosis of macrophages expressing a receptor of the CRP. Each ofmolecules of the C-reactive protein has their binding sites that can bespecifically bound to five phosphocholine groups.

In the chip for detection of the C-reactive protein in accordance withan illustrative embodiment, an end of the molecularly imprinted layer isarranged such that the five phosphocholine groups correspond tophosphocholine binding sites of respective molecules of the C-reactiveprotein.

In an illustrative embodiment, the fixing layer may be, but is notlimited to, a polymer layer, a self assembled monolayer, or mica, havingan azide or acetylene end group R¹ on the surface thereof. For example,the polymer layer, the self assembled monolayer, or the mica is fixed onthe metal substrate through a thiol group. The surface thereof may haveazide or acetylene group or may be modified through proper surfacetreatment to have azide or acetylene group. However, the presentdisclosure is not limited thereto.

Any polymer layer may be used if it is fixed on the metal substratethrough a functional group such as a thiol group, and the surfacethereof has an azide or acetylene group. For example, the polymer layermay be formed including, but is not limited to, polyacrylate,polymethacrylate, ethylene glycol amine, ethyleneglycolthiol, NHS(Nhydroxysuccinimide)-ethylene glycol, maleimide-ethylene glycol,polyethylene glycol amine, polyethylene glycol thiol, (NHS)-polyethyleneglycol, maleimide-polyethylene glycol, polymer based on carbohydratemonomer binding, and a combination thereof.

In an illustrative embodiment, the self assembled monolayer may beformed including, but is not limited to, HS—Y—R¹ (wherein R¹ is an azideor acetylene end group, and Y is a spacer group having a length of about0.1 nm to about 5 nm).

In an illustrative embodiment, the spacer group Y in HS—Y—R¹ forformation of the self assembled monolayer may be, but is not limited to,a hydrocarbon group or an oxyhydrocarbon group, which may include atleast one of double binding, triple binding, and an aromatic ring andhave about 1 to about 30 carbons. For example, the spacer group Y may bea hydrocarbon group or an oxyhydrocarbon group, which may include atleast one of double binding, triple binding, and an aromatic ring andhave about 1 to about 30 carbons, about 1 to about 25 carbons, about 1to about 20 carbons, or about 1 to about 15 carbons. However, thepresent disclosure is not limited thereto. In an illustrativeembodiment, the spacer group Y may be an alkylene group or alkyleneoxide(e.g., polyethyleneoxide and a polyprophyleneoxide group), which mayinclude at least one of double binding, triple binding, and an aromaticring and have about 1 to about 30 carbons, about 1 to about 25 carbons,about 1 to about 20 carbons, or about 1 to about 15 carbons. However,the present disclosure is not limited thereto. For example, the selfassembled monolayer may be formed including, but is not limited to,azido-C₁₋₃₀alkylene-1-thiol, acetylene-C₁₋₃₀alkylene-1-thiol, orpropargyloxy-C₁₋₃₀alkylene-1-thiol.

In an illustrative embodiment, a material for the metal substrate is notparticularly limited. Any material may be used if it is generally usedin the art to prepare a chip for detection of the C-reactive protein.For example, the metal substrate may be formed of, but is not limitedto, gold, silver, copper, or palladium, and more preferably, gold (Au).

In an illustrative embodiment, the molecularly imprinted layer may beformed including, but is not limited to, R²—X-phosphocholine (wherein R²is an azide or acetylene end group, and X is a spacer group having alength of about 0.1 nm to 5 nm). In an illustrative embodiment, themolecularly imprinted layer may be formed including, but is not limitedto, acetylene-C₁₋₃₀alkylene-phosphorylcholine,propargyloxy-C₁₋₃₀alkylene-phosphorylcholine, orazido-C₁₋₃₀alkylene-phosphorylcholine.

As described above, the azide or the acetylene end self assembledmonolayer is formed by fixing on the metal substrate through afunctional group capable of being bound to metal such as a thiol group.A molecularly imprinted layer is formed on the azide or the acetyleneend-self assembled monolayer through a click chemistry reaction. In thismanner, it is possible to prepare the chip for detection of theC-reactive protein, facilitating fixing a molecularly imprinted layer onthe metal substrate and improving a constant for binding to theC-reactive protein thereby providing an improved detection effect. Thechip for detection of the C-reactive protein in accordance with anillustrative embodiment exhibits a high constant for binding to theC-reactive protein. Accordingly, a low concentration of the C-reactiveprotein can also be detected, so that a concentration of the C-reactiveprotein in serums can also be measured by using surface Plasmonresonance. Since the click chemistry reaction is pH-independent, veryfast, and effective, it enables the molecularly imprinted layer to beeasily and effectively fixed onto the metal substrate. Accordingly, inan illustrative embodiment, it is possible to easily prepare the chipfor detection of the C-reactive protein without using an antibody.

A method for preparing a chip for detection of a C-reactive protein inaccordance with another aspect of an illustrative embodiment mayinclude:

forming a fixing layer having the azide or the acetylene end group R¹ ona metal substrate; mixing the C-reactive protein and a molecularlyimprinted material including R²—X-phosphocholine (wherein R² is azide oracetylene end group, and X is a spacer group having a length of about0.1 nm to about 5 nm) to obtain a mixture solution of the molecularlyimprinted material/the C-reactive protein; immersing the metalsubstrate, on which the fixing layer is formed, in the mixture solutionof the molecularly imprinted material/the C-reactive protein to obtain acomplex layer of the metal substrate/the fixing layer/the molecularlyimprinted material/the C-reactive protein through a click chemistryreaction between the end group R¹ of the fixing layer and R² included inR²—X-phosphocholine of the molecularly imprinted material; and removingthe C-reactive protein from the complex layer to obtain a molecularlyimprinted layer.

In an illustrative embodiment, if the end group R¹ of the fixing layeris an azide group, R² of the molecularly imprinted material may be anacetylene group. If the end group R¹ of the fixing layer is an acetylenegroup, R² of the molecularly imprinted material may be an azide group.However, the present disclosure is not limited thereto.

In an embodiment of the present disclosure, the fixing layer may be, butis not limited to, a polymer layer, a self assembled monolayer, or amica, having an azide or acetylene end group R¹ on the surface thereof.

The above-described preparing method may further include reactingpropagyl alcohol on a portion of the fixing layer, which has not beenbound to the molecularly imprinted material, after formation of thecomplex layer. However, the present disclosure is not limited thereto.

In an illustrative embodiment, the above-described preparing method mayfurther include pre-culturing the mixture solution of the molecularlyimprinted material/the C-reactive protein, prior to immersing the metalsubstrate on which the fixing layer is formed, such that fivephosphocholine groups derived from the molecularly imprinted materialare arranged to correspond to phophocholine binding sites of respectivemolecules of the C-reactive protein. However, the present disclosure isnot limited thereto.

In an illustrative embodiment, the molecularly imprinted material andthe C-reactive protein may be mixed with an equivalent ratio of about5:about 1. However, the present disclosure is not limited thereto. Forexample, each of molecules of the C-reactive protein has theirrespective phosphocholine binding sites capable of being bound to fivephosphocholine groups. Accordingly, if the molecularly imprintedmaterial and the C-reactive protein are mixed with the above-describedequivalent ratio for reaction or pre-culture, the molecularly imprintedmaterial is bound to the phosphocholine binding sites of the C-reactiveprotein, so that the phosphocoline groups of the molecularly imprintedmaterial can be arranged to correspond to the phosphocholine bindingsites of the C-reactive protein.

The descriptions of the chip for detection of the C-reactive protein inaccordance with an aspect of the present disclosure are applied to themethod for preparing a chip for detection of a C-reactive protein inaccordance with an illustrative embodiment. Thus, for convenience sake,overlapping descriptions in this regard are omitted herein.

Another aspect of the present disclosure provides a surface Plasmonresonance sensor, including the chip for detection of the C-reactiveprotein as stated as above. The descriptions of the chip for detectionof the C-reactive protein and the method for preparing the same areapplied to the surface Plasmon resonance sensor. For convenience sake,overlapping descriptions in this regard are omitted herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting and non-exhaustive embodiments will be described inconjunction with the accompanying drawings. Understanding that thesedrawings depict only several embodiments in accordance with thedisclosure and are, therefore, not to be intended to limit its scope,the disclosure will be described with specificity and detail through useof the accompanying drawings, in which:

FIG. 1 is a view schematically illustrating a process of binding betweena pentraxin protein and a reactive ligand according to an illustrativeembodiment;

FIG. 2 is a view for explanation of a method for preparing astyrene-phosphocholine ligand by steps according to an illustrativeembodiment;

FIG. 3 is NMR analysis results for a styrene-phosphocholine ligandprepared according to an illustrative working example;

FIG. 4 is a schematic view showing a method for preparing a molecularlyimprinted polymer using a styrene-phosphocholine ligand according to anexample;

FIG. 5 is a schematic view showing a method for preparing a molecularlyimprinted polystyrene film using a styrene-phosphocholine ligandaccording to an example;

FIG. 6 is a schematic view showing a method for preparing a molecularlyimprinted polymer using a styrene-phosphocholine ligand according to anexample;

FIG. 7 is a graph showing Langmuir plot to determine a binding constantfor a MIP in an example;

FIG. 8 shows analysis results for a selective binding force of a MIPusing BSA and CA;

FIG. 9 shows observation results for distribution of C-reactive proteinsbeing bound to a NIP, which is a control group of a MIP, by means of AFMin an example;

FIG. 10 is a microphotograph obtained by observing a polystyrene beadprepared according to an example by means of SEM;

FIG. 11(a) is a chemical formula showing a method for preparing11-azidoundecane-1-thiol (azido-thiol) and FIG. 11(b) is a chemicalformula showing a method for preparing 6-propargylhexylphosphorylcholine(propargyl-PC) in an example;

FIG. 12 is a process view showing a process for preparing a chip fordetection of a C-reactive protein in an illustrative embodiment;

FIG. 13 shows FT-IR spectrums for surface modification according to anexample;

FIG. 14 is a graph showing concentration-dependent binding of aC-reactive protein (CRP) to each of a control polymer (CM), and amolecularly non-imprinted monolayer, and a molecularly imprintedmonolayer (examples);

FIG. 15 is a graph for plotting binding of a C-reactive protein to eachof a molecularly non-imprinted monolayer and a molecularly imprintedmonolayer according to the Langmuir isotherm equation;

FIG. 16 is a graph showing an effect of free phosphocholine in bindingof a C-reactive protein to each of a molecularly non-imprinted monolayerand a molecularly imprinted monolayer (examples);

FIG. 17 is a graph showing binding of a C-reactive protein in serums toeach of a control polymer, a molecularly non-imprinted monolayer, and amolecularly imprinted monolayer (examples); and

FIG. 18 is a graph showing an effect of free phosphocholine in bindingof a C-reactive protein in a complex matrix in an example.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, illustrative embodiments will be described in more detailwith reference to illustrative working examples. The illustrativeworking examples are merely exemplary, and the scope of the presentdisclosure should not be construed as being limited to the illustrativeworking examples.

Example 1 Preparation of a Styrene-Phosphocholine Ligand

A process for preparing a styrene-phosphocholine ligand was describedbelow with reference to FIG. 2. The products presented in FIG. 2 havebeen identified through NMR.

12-hydroxydecanoic acid and potassium carbonate were added into 100 mlof N,N′-dimethylformamide and stirred at a room temperature for about 10minutes. Thereafter, 4-vinylbenzyl chloride was added thereinto andstirred while being heated at less than about 80° C. After the reactionwas finished, N,N′-dimethylformamide was removed through distillationunder reduced pressure. The reacting material was dissolved indichloromethane and then washed with water. Dichloromethane layers werecollected and dried with a magnesium sulfate. From the drieddichloromethane, the magnesium sulfate was removed by a depressurizationfilter, and dichloromethane was removed through distillation underreduced pressure. Thereafter, column chromatography purification usingan eluent of hexane and ethyl acetate with a ratio of 3:1 was performed(yield=79%).

₁H NMR (CDCl₃ 400 MHz): δ (ppm) 7.41 (m, 4H), 6.75 (m, 1H), 5.78 (dd,J₁=13.6, J₂=4.0, ₁H), 5.29 (dd, J1=6.8, J2=4.0, 1H), 5.10 (d, J=7.2,2H), 3.63 (t, J=4.4, 2H), 2.₃7 (t, J=4.8, 2H), 1.64 (m, 4H), 1.27 (br s,14H)

₁₃C NMR (CDCl3 100 MHz): δ (ppm) 173.74, 137.90, 137.52, 136.47, 136.40,136.35, 135.62, 128.77, 128.47, 127.60, 126.36, 126.05, 126.01, 114.39,114.30, 65.97, 65.83, 63.11, 34.35, 32.81, 29.56, 29.49, 29.41, 29.24,29.12, 25.74, 24.96

LRMS (ESI+) m/z: 355 (M+Na)

FT-IR (neat) ν (cm⁻¹): 3328 (br w), 2914 (s), 2848 (s), 1728 (s), 1462(m), 1386 (w), 1359 (w), 1332 (m), 1316 (w), 1256 (m), 1195 (s), 1107(m), 1057 (m), 1042 (m), 1008 (m), 989 (m), 962 (m), 899 (m), 830 (w),850 (w), 804 (m), 728 (m)

1.5 g of 4-Vinylbenzyl-12-hydroxyl dodecanoate (496 mmol) and 2.09 ml oftriethylamine (14.9 mmol) were added into 20 ml of dichloromethane(amylene stabillized) and stirred while being cooled at 0° C. on anice-bath. 0.684 ml of 2-chloro-2-oxo-1,3,2-dioxaphospholane (7.44 mmol)was added into the reacting material and stirred at 0° C. for about 1hour. Thereafter, the ice-bath was removed, and the reacting materialwas reacted at a room temperature. After the reaction was finished,dichloromethane was removed through distillation under reduced pressure.The reacting material was dissolved in chloroform and undissolved solidswere removed by means of a depressurization filter. Columnchromatography purification using an eluent of hexane and ethyl acetatewith a ratio of 3:1 was performed (since the intermediate compound waseasily denatured, a next follow-up reaction was proceeded with afteridentification of ₁H NMR). 0.864 g of12-(2-Oxo-2λ5-[1,3,2]dioxaphospholan-2-yloxy)-dodecanoic acid4-vinyl-benzyl ester (1.97 mmol) were added into a pressure bottletogether with an acetonitrile anhydrous and cooled to −20° C. 0.55 ml oftrimethylamine (5.92 mmol) was added into the cooled pressure bottle andheated at less than 60° C. with stirring.

After the reaction was finished, acetonitrile was removed throughdistillation under reduced pressure. Column chromatography purificationusing an eluent of chloroform, methanol, and water with a ratio of65:25:4 was performed (yield=24%).

₁H NMR (CDCl₃ 400 MHz): δ (ppm) 7.42 (m, 4H), 6.73 (m, 1H), 5.77 (dd,J₁=13.6, J₂=4.0, 1H), 5.27 (dd, J1=6.8, J2=4.0, 1H), 5.08 (d, J=7.2,2H), 4.26 (br s, 2H), 3.84 (m, 4H), 3.41 (s, 9H), 2.36 (m, 2H), 1.62(_(m), 4H), 1.24 (br s, 14H)

₁₃C NMR (CD_(C)l3 100 M_(H)z): δ (ppm): 173.68, 137.87, 137.50, 136.46,136.39, 136.33, 135.61, 128.77, 128.44, 127.57, 126.35, 126.01, 125.99,114.39, 114.30, 66.25, 65.95, 65.81, 65.66, 65.60, 59.20, 54.29, 34.32,31.07, 31.00, 29.70, 29.64, 29.53, 29.49, 29.32, 29.16, 25.92, 24.96

LRMS (ESI+) m/z: 520 (M+Na)

FT-IR (neat) ν (cm⁻¹): 3367 (br w), 2923 (s), 2852 (m), 1732 (s), 1483(w), 1380 (w), 1232 (s), 1164 (s), 1057 (s), 986 (s), 966 (s), 909 (s),874 (w), 825 (s), 796 (s), 763 (s), 713 (s), 505 (s)

The styrene-phosphocholine ligand (12-(4-vinylbenzyl)oxycarbonyl dodecylphosphocholine) that has been prepared as described above was identifiedas a final product by means of NMR (Nuclear Magnetic Resonance; Avance400; the Bruker company). FIG. 4 shows results of the NMR analysis.

Example 2 Preparation of a Polystyrene Film, in which aStyrene-Phophocholine Ligand is Molecularly Imprinted

A method for preparing a molecularly imprinted polymer according to anillustrative embodiment is described below with reference to FIG. 5.

Total 1 ml of styrene/DVB (divinylbenzene) as a crosslink agent, and DBK(dibenzylketone) as an initiator, were added to 10 ml of a standardphosphate buffer solution (pH 7.0) with a ratio ofstyrene:DVB:DBK=9:1:1. Thereafter, a C-reactive protein and astyrene-phosphocholine ligand, which have been reacted together inadvance, were added thereto with a molar ratio of 1:5. Since theC-reactive protein and the styrene-phosphocholine ligand were stronglybound to each other via calcium ions, they have been reacted in advancein a binding buffer solution (0.1M Tris, 5 mM CaCl₂, 150 mM NaCl, and pH8.0) thereby inducing photo-polymerization thereafter. With respect toconditions for the polymerization reaction, photo-polymerization wasperformed at 37° C. for 2 hours such that a monomer(styrene-phophocholine ligand) is positioned on a surface of thecrosslink agent layer in the state that the monomer is bound to theC-reactive protein (here, since the ligand has both a hydrophilic groupand a hydrophobic group, the hydrophilic group is directed toward awater phase, and the hydrophobic group is directed toward a surface ofstyrene/DVB). Subsequently, photo-polymerization was performed at 90° C.for 2 hours such that a hard polystyrene polymer can be formed. Afterthe polymerization reaction was finished, in order to remove theC-reactive protein, the reacting material was washed with an elutionbuffer solution (0.1 M Tris, 10 mM EDTA, 150 mM NaCl, pH 8.0) twice.

Experimental Example 1 Measurement of a Binding Force of a C-ReactiveProtein to a Molecularly Imprinted Polymer

A binding force of a C-reactive protein to a molecularly imprintedpolymer was analyzed by detecting a C-reactive protein through theBradford assay method and an immunoassay method. In this case, inaddition to a molecularly imprinted polymer (MIP), a molecularlynon-imprinted polymer (NIP) and a polystyrene polymer (control polymer;CP) were prepared to analyze a molecularly imprinting effect. Themolecularly non-imprinted polymer was prepared by using a reactiveligand without molecularly imprinting of CRP. The polystyrene polymerwas prepared without using a reactive ligand and CRP.

(1) Bradford Assay Method

5 μg of a CRP was added to the prepared molecularly imprinted polymerand reacted at 0° C. for 30 minutes. In order to remove anon-specifically bound protein, the reacting material was washed threetimes with PBS or repeatedly washed three times with a TBST buffersolution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1% Tween20. Thereafter, 2 ml of an elution buffer solution was added thereto,and the reacting material was kept at a room temperature for 15 minutes.In order to quantify an amount of the protein in the elution buffersolution, 1 ml of a Bradford reagent (the Sigma company) was added to 1ml of an eluted protein solution, and the solution was kept at a roomtemperature for 10 minutes. Thereafter, absorbance was measured at 595nm. In order to prepare a standard quantification curve, 0, 0.5, 1, 1.5,and 2 μg/ml of BSA solutions were prepared. 1 ml of the Bradford reagentwas added to 1 ml of each of the BSA solutions, and the solution waskept at a room temperature for 10 minutes. Thereafter, absorbance wasmeasured at 595 nm (Varian-300 spectrophotometer).

(2) Immunoassay Method

5 μg of a CRP was added to the prepared molecularly imprinted polymerand reacted at 0° C. for 30 minutes. In order to remove anon-specifically bound protein, the reacting material was washed threetimes with PBS or repeatedly washed three times with a TBST buffersolution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1% Tween20. Thereafter, 10 μg of anti CRP mouse monoclonal antibody (the Sigmacompany) was added thereto, and the reacting material was reacted at 0°C. for 30 minutes. In order to remove a non-specifically bound antibody,the reacting material was washed three times with PBS or repeatedlywashed three times with a TBST buffer solution (0.1 M Tris, 150 mM NaCl,0.1% Tween 20) containing 0.1% Tween 20. Thereafter, an anti mouse IgG(the Sigma company), to which alkaline phosphatase was bound, wasdiluted with a ratio of 1:30,000 and added thereto. Thereafter, thereacting material was reacted at 0° C. for 30 minutes. In order toremove a non-specifically bound antibody, the reacting material waswashed three times with PBS or repeatedly washed three times with a TBSTbuffer solution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1%Tween 20. Thereafter, 1 ml of an NPP solution (p-nitrophenolphosphate;the Sigma company), which is a substrate of alkali phosphatase, wasadded thereto, and the reacting material was kept at a room temperaturefor 10 minutes. Thereafter, absorbance was measured at 405 nm(Varian-300 spectrophotometer).

TABLE 1 Effect of molecularly imprinting a C-reactive protein Assaymethod/Washing buffer Bradford assay (ug) Immunoassay (O.D) Sample PBSTBST PBS TBST CP 0.45 ± 0.0073 0.13 ± 0.021 — — NIP 1.4 ± 0.051 0.27 ±0.041 0.48 ± 0.061 0.024 ± 0.0051 MIP 3.0 ± 0.71  2.6 ± 0.17 1.3 ± 0.180.19 ± 0.061 IF 2.1 9.8 2.6 8.1

Table 1 shows comparison of molecularly imprinting factors (imprintingfactor; IF) of C-reactive proteins of a MIP and a NIP. An amount of aC-reactive protein bound to the MIP was higher 8.1 or 9.8 times than anamount of a C-reactive protein bound to the NIP depending on a testmethod. It is understood that the C-reactive protein on the NIP waseluted during the washing process, whereas the C-reactive protein on theMIP was not eluted and remains.

Experimental Example 2 Determination of a Binding Constant to a MIP

FIG. 7 is a graph using the Langmuir isotherm equation. Based on slopeand a y-intercept value, a MIP-C-reactive protein binding constant K_(A)is 3.0×10⁹/M. Below is the Langmuir isotherm equation:

$\frac{C}{R} = {\frac{C}{R_{\max}} + {\frac{1}{R_{\max}} \times \frac{1}{K_{A}}}}$

R_(max) can be calculated from a reciprocal number of the slope, andK_(A) can be calculated from the y-intercept.

Following table 2 shows CRP binding constants that have been reporteduntil now. The detection method using a molecularly imprinted polymer inan illustrative embodiment exhibits a binding constant value higherabout three degrees than that in detection methods using a conventionalanti C-reactive protein antibody.

TABLE 2 Comparison of binding constants in the detection method of anillustrative embodiment and a conventional detection method BindingDetection constant Receptor method (K_(A), M⁻¹) Reference Anti-CRPantibody SPR 1.4 × 10⁶ 1 (immobilized) Anti-CRP antibody fiber-optic 3.8× 10⁸ 2 (immobilized) detection Exposed PC on SPR 9.9 × 10⁶ 3 supportedlipid monolayers^(a) PC-appended ELISA 7.1 × 10⁶ 4 supramolecularassembly The MIP in this MSIA 3.0 × 10⁹ — study 1. M. H. F. Meyer, M.Hartmann and M. Keusgen, Biosens. Bioelectron., 2006, 21, 1987 2. C.Chou, H. Y. Hsu, H. T. Wu, K. Y. Tseng, A. Chiou, C. J. Yu, Z. Y. Leeand T. S. Chan, J. Biomed. Opt., 2007, 12, 024025 3. S. F. Sui, Y. T.Sun and L. Z. Mi, Biophys. J., 1999, 76, 333; 4. B. A. Rosenzweig, N. T.Ross, D. M. Tagore, J. Jayawickramarajah, I. Saraogi and A. D. Hamilton,J. Am. Chem. Soc., 2009, 131, 5020.

Experimental Example 3 Analysis of a Selective Binding Force of a MIP

In order to identify a specific binding force of a MIP to a CRP, 5 μg ofbovine serum albumin (BSA) and 5 μg of carbonic anhydrase (CA) wereadded into each of the MIP and the NIP. The binding force was analyzedin the same manner as described in Experimental Example 1.

FIG. 8 shows binding forces of the MIP and the NIP to BSA and CA. Asshown in FIG. 8, the PS and the NIP exhibit a low binding force of lessthan 0.5 μg to the CRP, BSA, and CA. However, the MIP exhibits a higherbinding force of more than 2.5 μg only to the C-reactive protein. Itshows that the MIP was specifically bound to the CRP.

Experimental Example 4 AFM Analysis

FIG. 9 shows observance results for distribution of C-reactive proteinsbeing bound to a MIP and a NIP by means of an atomic force microscope(AFM; the Vecco company; model code: D-V). Upon observation, the heightof a side surface of the protein on the MIP is relatively regular, whilethe protein on the NIP is partially agglutinated or bound with its sidesurface having irregular height. In case of the molecularly imprintedprotein, pentamers are simultaneously bound thereby exhibiting a planeand regular height of the side surface. However, on the surface of themolecularly non-imprinted polymer, binding through only one ligand ispossible. Accordingly, the protein binding shape and the proteindistribution were irregular, which were different from those in the MIP.

Example 3 Preparation of a Polystyrene Bead, in which a FunctionalMonomer is Molecularly Imprinted

A process for preparing a molecularly imprinted polystyrene bead was thesame as Example 1. However, in order to disperse styrene/DVB on anaqueous solution, 0.1% Brij 78 was added as a dispersant, and then,photo-polymerization was performed. Under the same conditions as thosein Example 2, the 0.1% Brij 78 dispersant was added, such thatstyrene/DVB was dispersed in a bead form through vigorous stirring.During the photo-polymerization, styrene/DVB was continuously dispersedthrough stirring.

FIG. 10 shows results of observation of the prepared polystyrene bead bymeans of an SEM. The size of the bead is in a range of 1.2 μm to 1.8 μm.A molecularly imprinting factor is 3.6. A detection limit, which can beidentified by the Bradford assay method, is 0.3±0.1 μg/ml. Accordingly,CRP detection is possible at a concentration below a blood concentrationrange. Upon measurement of surface conductivity, the surfaceconductivity of the polystyrene bead, to which no ligand is bound, is0.0767 mS/cm. The surface conductivity of the bead, in which a ligand ismolecularly imprinted, is 9.98 mS/cm. Since one end of the reactiveligand has a choline group and a phosphate group, they exhibit + and −charges. For this reason, the surface conductivity of the bead in whicha ligand is molecularly imprinted appears to have increased, compared tothat of the polystyrene bead to which no ligand is bound.

Example 4 Synthesis of 11-azidoundecane-1-thiol

All reagents for chemical synthesis were purchased from Sigma-AldrichChemical and used without additional purification. Human plasma and allbuffer components were also purchased from Sigma-Aldrich Chemical. For aCRP-negative human serum (N-serum), components of the CRPA Latex TestSet purchased from Cenogenics (Morganville, USA) were used. Phosphatebuffered saline (PBS) and HyClone(R) were purchased from ThermoScientific (Rochester, USA). 1H (400 MHz) and 13C (100 MHz) NMRspectrums were measured by using Bruker Spectrospin 400.

11-bromo-1-undecanol (1 g, 3.98 mmol), sodium azide (285 mg, 4.38 mmol),and potassium iodide were dissolved in ethanol and refluxed for 20hours. In the solution, a solvent was removed under reduced pressure,and a residue was dissolved in diethylether. The residue mixturedissolved in the ether was washed with water and dried on an anhydrousmagnesium sulfate. The solvent was removed under reduced pressure. Anon-purified product produced by the removal of the solvent was purifiedon silica gel (R_(f)=0.3, hexane:EtOAc=5:1) through columnchromatography to obtain a compound 1 (868 mg, 102.2%).

IR: 3332, 2924, 2853, 2091

¹H NMR (400 MHz, CDCl₃) δ 3.57 (t, 2H, OCH₂), 3.32 (s, 1H, OH), 3.25 (t,2H, N₃CH₂), 1.57 (m, 4H, HOCH₂CH₂ (CH₂)₇CH₂), 1.28 (m, 14H,HOCH₂CH₂(CH₂)₇)

¹³C NMR (100 MHz, CDCl₃) δ62.4, 51.4, 32.6, 29.5, 29.4, 29.1, 28.8,26.7, 25.7

Exact mass calcd for C₁₁H₂₃N₃O: 213.18. found: 236 [M+Na]⁺.

The compound 1 (868 mg, 4.09 mmol), methanesulfonyl chloride (1.26 g,11.0 mmol), and triethylamine (2.44 g, 24.1 mmol) were dissolved in THF.The reaction mixture was stirred at a room temperature for 2 hours.After addition of ice water, an organic phase in the mixture wasseparated from an aqueous phase in the mixture. The aqueous phase wasextracted twice by diethylether. The organic phase was washed with 1 MHCl, deionized water, and saturated sodium bicarbonate. After theorganic phase was dried on an anhydrous magnesium sulfate, a solvent inthe organic phase was removed under reduced pressure. A resultantnon-purified product was purified on silica gel (R_(f)=0.4,hexane:EtOAc=5:1) through column chromatography to obtain a compound 2(1.17 g, 98.2%).

IR: 2925, 2854, 2092, 1180

¹H NMR (400 MHz, CDCl₃) δ 4.21 (t, 2H, OCH₂), 3.26 (t. CH₂N₃), 3.00 (s,3H, CH₃S), 1.74 (m, 2H, OCH₂CH₂), 1.59 (m, 2H, CH₂CH₂N₃) 1.39-1.18 (m,14H, OCH₂CH₂(CH₂)₇)

¹³C NMR (100 MHz, CDCl₃) δ70.4, 51.4, 37.1, 29.4, 29.3, 29.11, 29.10,29.0, 28.8, 26.7, 25.4

Exact mass calcd for C₁₂H₂₅N₃O₃S: 291.16. found: 314 [M+Na]⁺.

The compound 2 (1.17 mg, 4.01 mmol) and potassium thioacetate (917 mg,8.03 mmol) were dissolved in 90 mL of DMF. The reaction mixture wasstirred at a room temperature for 1 hour. In the reaction mixture, asolvent was removed under reduced pressure, and a residue was dissolvedin diethyl ether. An organic phase formed of the residue was washed withwater and dried on an anhydrous magnesium sulfate. The solvent wasremoved under reduced pressure. A resultant non-purified product waspurified on silica gel (R_(f=0.7), hexane:EtOAc=9:1) through columnchromatography to obtain a compound 3 (831 mg, 76.4%).

IR: 2924, 2853, 2092, 1690

¹H NMR (400 MHz, CDCl₃) δ 3.25 (t, 2H, CH₂N₃), 2.85 (t, 2H, SCH₂), 2.31(s, 3H, CH₂CO), 1.57 (m, 4H, SCH₂CH₂ (CH₂)₇CH₂), 1.35-1.27 (m, 14H,SCH₂CH₂(CH₂)₇)

¹³C NMR (100 MHz, CDCl₃) δ195.8, 51.4, 30.6, 29.5, 29.4, 29.15, 29.11,28.86, 28.81, 26.7

Exact mass calcd for C₁₃H₂₅N₃OS: 271.17. found: 294 [M+Na]⁺.

The compound 3 (588 mg, 2.17 mmol) was dissolved in 40 ml of methanoland 2 ml of concentrated HCl. The reaction mixture was stirred for 3hours and quenched with water. In the reaction mixture, an aqueous phasewas extracted twice by diethyl ether. In the reaction mixture, anorganic phase was washed with water and dried on an anhydrous magnesiumsulfate. A solvent of the organic phase was removed under reducedpressure. A resultant non-purified product was purified on silica gel(R_(f)=0.8, hexane:EtOAc=9:1) through column chromatography to obtain acompound 4 (471 mg, 94.6%).

IR: 2923, 2852, 2090

¹H NMR (400 MHz, CDCl₃) δ 3.25 (t, 2H, CH₂N₃), 2.51 (q, 2H, SCH₂), 1.60(m, 4H, SCH₂CH₂(CH₂)₇CH₂), 1.35-1.28 (m, 15H, HSCH₂CH₂ (CH₂)₇)

¹³C NMR (100 MHz, CDCl₃) δ51.4, 34.1, 29.5, 29.1, 29.0, 29.8, 28.4,26.7, 24.6.

Example 5 Synthesis of 6-propagylhexylphosphorylcholine(propagyl-phosphorylcholine)

A solution of 1,6-hexandiol (3 g, 25.4 mmol) in DMF (20 ml) is dropwisedinto a suspension of sodium hydride (1.52 g, 38.1 mmol) in the DMF (20ml) on an ice bath, and stirred for 30 minutes under the ice bathcondition. Propagyl bromide (5.7 g, 38.1 mmol) in the DMF (20 ml) wasadded to the mixture, and then, stirred at a room temperature for 20hours. The solvent was removed under reduced pressure. A resultantnon-purified product was dissolved in diethylether. The mixture waswashed with water, dried on an anhydrous magnesium sulfate, and purifiedon silica gel (R_(f)=0.5, hexane/EtOAc=1:1) through columnchromatography to obtain a compound 5 (1.80 g, 45.4%).

IR: 3373, 3292, 2933, 2858, 1093

¹H NMR (400 MHz, CDCl₃) δ4.13 (s, 2H, HCCCH2O), 3.59 (t, 2H, CH₂OH),3.52 (t, 2H, CH₂OCH₂), 3.03 (s, 1H, CH₂OH), 2.48 (s, 1H, HCCCH₂O), 1.58(m, 4H, OCH₂CH₂CH₂CH₂CH₂), 1.38 (m, 4H, OCH₂CH₂CH₂CH₂)

¹³C NMR (100 MHz, CDCl₃) δ 79.8, 74.3, 70.0, 62.3, 57.9, 32.5, 29.3,25.8, 25.5

Exact mass calcd for C₉H₁₆O₂: 156.12. found: 179 [M+Na]⁺.

The compound 5 (600 mg, 3.84 mmol),2-chloro-1,3,2-dioxaphosphoran2-oxide (1.09 g, 7.68 mmol), andtriethylamine (777 mg, 7.68 mmol) were dissolved in 30 ml of DCM. Thereaction mixture was stirred at a room temperature for 72 hours under adarkroom condition. The solvent was removed under reduced pressure. Aresultant non-purified product was purified on silica gel (R_(f)=0.3,hexane/EtOAc=1:4) through column chromatography to obtain a compound 6(700 mg, 69.5%).

The compound 6 (700 mg, 2.66 mmol) and trimethylamine (1.57 g, 26.6mmol) were dissolved in 8 ml of acetonitrile. The reaction mixture wasstirred at 60° C. for 20 hours. The solvent was removed under reducedpressure. A resultant non-purified product was purified on silica gel(chloroform:methanol is 2:1; and chloroform:methanol:water is 50:50:4;R_(f)=0.2) through column chromatography. The solvent was removed underreduced pressure. The residue was dissolved in anhydrous chloroform andfiltered to obtain a compound 7 (411 mg, 48.1%).

IR: 3350, 3296, 2936, 2860, 1086, 1059

¹H NMR (400 MHz, CD₃OD) δ 4.28 (m, 2H, POCH₂CH₂N), 4.15 (d, 2H,HCCCH₂O), 3.90 (q, 2H, CH₂CH₂CH₂OP), 3.69 (m, 2H, POCH₂CH₂N+), 3.54 (t,2H, HCCCH₂OCH₂), 3.27 (s, 9H, N⁺(CH₃)₃), 2.85 (t, 1H, HCCCH2O), 1.64 (m,4H, OCH₂CH₂CH₂CH₂CH₂), 1.44 (m, 4H, OCH₂CH₂CH₂CH₂)

¹³C NMR (100 MHz, CDCl₃) δ79.7, 74.8, 69.6, 65.5, 59.0, 57.4, 53.5,30.4, 29.2, 25.6, 25.3

Exact mass calcd for C₁₄H₂₈NO₅P: 321.17. found: 322 [M+H]).

Example 6 Surface Modification of an SPR Chip

FIG. 12 shows a whole scheme for surface modification of an SPR chip. Inorder to prepare a self assembled monolayer (SAM) having anazide(N₃)-terminated compound, an SPR chip (15 mm×15 mm×10 mm) wasimmersed in 2 mM (20 ml) of an ethanol solution of11-azidoundecane-1-thiol at a room temperature for 12 hours (FIG. 12).The substrate was thoroughly washed with ethanol several times, andthen, dried under N₂ gas. For pre-test for a click reaction, a goldsubstrate (5 mm×5 mm×1 mm) was coated with 11-azidoundecane-1-thiol inthe same manner as described above, and placed in a reaction mixturecontaining 1 μmol of propagyl-phosphorylcholine in 1 ml of PBS (FIG.12), 0.5 μmol of copper sulfate (II).pentahydrate, and 1 μmol of asodium ascorbic acid at 4° C. for 16 hours.

In order to prepare a molecularly imprinted monolayer (MIM), 3.32 nmolof propagyl phosphorylcholine (PC) was pre-cultured on an ice bath for30 minutes, with 0.66 nmol of a C-reactive protein (CRP) (molar ratio of5:1 for propagyl phosphorylcholine: CRP) in 1 ml of a binding buffer(0.1 M Tris/HCl, 150 mM NaCl, 5 mM CaCl₂, pH 8.0). A 1,3-dipolarcycloaddition reaction, which is one of click chemistry reactions, wasperformed by placing the SPR chip coated with the self assembledmonolayer of 11-azidoundecane-1-thiol in 20 ml of a PBS buffer solutioncontaining 1.66 nmol of copper sulfate (II).hydrate, 3.32 nmol of asodium ascorbate, and the propagyl phosphorylcholine-CRP complexpre-cultured at 4° C. for 16 hours. The molar ratio of propagylphosphorylcholine:copper sulfate:a sodium ascorbate was 1:0.5:1.

In order to prepare a molecularly non-imprinted monolayer, all stepswere carried out as those for the molecularly imprinted monolayer,except that no CRP was added to the reaction mixture. After the reactionwas finished, the surface was thoroughly washed with cool PBS and driedunder N₂ gas.

In order to block an azide end that has not been involved in the clickreaction, propagyl alcohol for another click reaction was added to thesubstrate. The substrate was immersed in 20 ml of PBS containing 20 μmolof propagyl alcohol, 10 μmol of copper sulfate (II).hexahydrate, and 20μmol of a sodium ascorbate at a room temperature for 5 hours.

In order to prepare a control polymer (CM), the SPR chip coated with theN₃-terminated self assembled monolayer was directly modified by propagylalcohol through a click reaction. A surface, which was not coated withpropagyl phosphorylcholine, was obtained.

In order to remove the CRP from the molecularly imprinted monolayer, thesurface-modified SPR chip and the gold substrate were immersed in anelution buffer (0.1 M Tris/HCl, 10 mM EDTA, 150 mM NaCl, pH 8.0) at aroom temperature for 30 minutes. Finally, the substrate was thoroughlywashed with deionized water, acetone, and ethanol, and dried under N₂gas.

Analysis of Surface Characteristics of an SPR Chip by Means of FT-IR

An FT-IR/ATR spectrum was acquired in a single reflection mode by usingthe Thermo Nicolet iS10 FT-IR spectrometer equipped with a smartaperture gazing angle (SAGA). IR spectrums of a gold surface, which hasbeen functionalized in each of the above-described reaction steps, wereobtained in a range of 650 cm⁻¹-4000 cm⁻¹. All the spectra wereequalized through 512 scan and reported in a penetration mode to a cleangold surface.

Analysis of CRP Binding Through a Surface Plasmon Resonance (SPR)Research

CRP binding to each substrate was measured by SPR LAB (the K-MACcompany). Dilution and elution of the sample were carried out with aflow rate of 20 ml/min at 25° C. by means of a binding buffer. Bindingof the CRP onto the SPR chip was carried out by one dose of injection of300 of the protein solution of 100 pM to 400 nM in the binding buffer. AN-serum was diluted to be a 2% solution in a binding buffer. BSA wasprepared to be a 1% solution in PBS. In the binding buffer, 10 mM offree phosphocholine (f-phosphocholine) was used to identifyphosphorylcholine specific binding of the CRP. The protein binding wasrecorded with a resonance angle and reported with incidence angle shift(Δ°).

Analysis of Surface Characteristics of an SPR Sensor

In order to introduce an azide group onto a surface of the goldsubstrate for continuous click chemistry, the gold substrate wasimmersed in 2 mM of an ethanol solution of 11-azidoundecane-1-thiol,which was azide-containing thiol, at a room temperature for 12 hours.Characteristics of a self assembled monolayer obtained from theimmersion were analyzed by means of FT-IR/ATS. In the IR spectrums,characteristic peaks of the self assembled monolayer were observed at2850 cm⁻¹ (symmetric methyl C—H stretching), 2930 cm⁻¹ (non-symmetricmethyl C—H stretching), and 2090 cm⁻¹ (non-symmetric N₃ stretching mode)(FIG. 13b ).

In order to identify that the click chemistry is caused by propagylphosphorylcholine, an excess molar concentration of propagylphosphorylcholine (1 mM) was added to the reaction mixture containingthe gold substrate coated with 11-azidoundecane-1-thiol. FIG. 13c showsan IR spectrum of the gold substrate, in which propagylphosphorylcholine at a 4-site of triazole was substituted with an excessamount of propagyl phosphorylcholine. In the spectrum, the non-symmetricN₃ stretching peak has disappeared at 2090 cm⁻¹. This shows thatmounting of propagyl phosphorylcholine onto the surface was accomplishedby successful coupling between an azide group and an acetylene group.

In synthesis of a molecularly non-imprinted monolayer (Click-NIM) and amolecularly imprinted monolayer (Click-MIM), a molar concentration ofpropagyl phosphorylcholine was 166 nM. In the IR spectrum, thenon-symmetric N₃ stretching peak has not disappeared at 2090 cm⁻¹ (FIG.13d and FIG. 13e ). This shows that the free N₃ group remains on thesurface.

In order to quench an activity surface of the free N₃ group, propagylalcohol, which is another agent for blocking click reaction, is added.After the blocking reaction, IR spectrums (FIGS. 13f, 13g, and 13h ) forthe control polymer (Blocking-CM), the molecularly non-imprintedmonolayer (Blocking-NIMM), and the molecularly imprinted monolayer(Blocking-MIM) show that the N₃ group is completely protected bypropagyl alcohol. It is identified that the non-symmetric N₃ stretchingpeak has disappeared completely at 2090 CM⁻¹. The peaks at 2850 CM⁻¹(symmetric methyl C—H stretching) and 2930 CM⁻¹ (non-symmetric methylC—H stretching) were preserved even after the click reaction.

Analysis of Binding Characteristics of a CRP to a Molecularly ImprintedMonolayer

The SPR technology relates to an optical method to measure a refractiveindex of a very thin layer of a material absorbed to a metal. Degree ofbinding between a target molecule in a solution and a molecular receptorfixed onto the chip surface was easily observed and quantified bymonitoring reflectivity variation. One of advantages of the SPRtechnology relates to exhibiting high sensitivity to the target moleculeor the receptor molecule without using labeling. The SPR was used toresearch binding of the CRP onto the modified gold surface.

In the system used in an illustrative embodiment, i.e., SPR LAB (theK-MAC company), reflectivity was measured with a function for anincidence angle of a light beam. Angle variation, to a coated goldsensor surface, was measured with about 1 milli degree of resolutioncorresponding to ˜10 μg/mm² of a protein bound onto the coated surface.Analysis for the SPR system was measured by variation of a resonanceangle, which was related to an amount of an analyte bound onto thesensor surface. A difference in a normal-state SPR signal is defined byincidence angle shift (Δ°) and proportional to a mass of an absorbedprotein (0.1°≈1 ng/mm²). The incidence angle shift was calculated by anincidence angle after washing with a buffer for 5 minutes and anincidence angle prior to injection of a protein.

Through an X-ray crystallography research, it was identified that adiameter of the CRP was 12 nm, and a binding region of the SPR chip was28.9 mm. Accordingly, based on 115 kDa of the molecular weight of theCRP, the whole binding ability of the SPR chip calculated to 49 ng (=1.7ng/mm²).

FIG. 14 shows an SPR signal by binding of a C-reactive protein (CRP) toa gold substrate, a control polymer (CM), a molecularly non-imprintedmonolayer (NIM), and a molecularly imprinted monolayer (MIM),respectively. In case of the control polymer, no CRP binding wasobserved. In case of the molecularly imprinted monolayer (MIM) with 10nM of the CRP concentration, binding of the CRP was almost half incomparison with that in the saturated state. Binding in case of 100 nMof the CRP concentration is almost same with that in the saturatedstate. In case of the molecularly non-imprinted monolayer (NIM), no CRPbinding was observed up to 100 nM of the CRP concentration. CRP bindingrapidly increases from 200 nM to 400 nM. The concentration-dependentbinding of the CRP to each of the molecularly non-imprinted monolayerand the molecularly imprinted monolayer shows that phosphorylcholine hasbeen successfully introduced onto the gold surface through clickchemistry, and provides a binding ability of the CRP to each of themolecularly non-imprinted monolayer and the molecularly imprintedmonolayer. If there is no non-specific binding in binding between themolecularly non-imprinted monolayer and the CRP, it is likely that atotal CRP binding ability (the number of binding sites) in themolecularly non-imprinted monolayer is greater than that in themolecularly imprinted monolayer.

A binding constant and a maximum binding ability (R_(max)) between theCRP and each of the molecularly non-imprinted monolayer and themolecularly imprinted monolayer were calculated by using a Langmuirabsorption model. The correlation between CRP concentration and angleshift as shown FIG. 14 is expressed with a following reaction formula,i.e., a modified Langmuir equation model:

$\begin{matrix}{\frac{C}{R} = {\frac{C}{R_{\max}} + \frac{1}{R_{\max} \cdot K_{A}}}} & (1)\end{matrix}$

Here, R is incidence angle shift caused by CRP binding in anequilibriuma state, C is CRP concentration, R_(max) is angle shift whenC is infinite, and K_(A) is an apparent binding constant.

As a result of plotting according to the above equation (1), a graph ina straight line was obtained, and a proportion of slope to the interceptwas K_(A) (FIG. 15). A K_(A) value for the molecularly imprintedmonolayer and the CRP was determined to be 1.33×10⁸ M⁻¹. However,binding between the molecularly non-imprinted monolayer and the CRP wasnot plotted by the Langmuir equation, and C and C/R are in the ininverse proportion to each other. Although an exact cause for therelation of inverse proportion is not provided herein, it is assumedthat the relation of inverse proportion can be explained in non-specificbinding of the CRP to the molecularly non-imprinted monolayer.

Upon calculation, a maximum coverage of the CRP on the SPR chip was 49ng. However, when 100 nM of the CRP was injected into a sample tube(buffer volume of 100 nM=12.0 ng/ml and chip surface=51.5 ml), total 618ng of the CRP was injected onto the chip surface during driving of thesample. Since CRP binding to the NIM started in concentration between100 nM and 200 nM of the CRP, the surface can be covered completely bythe CRP at an initial stage of the CRP binding. Thereafter,protein-protein attraction causes sudden non-specific binding of the CRPto the NIM in addition to PC-specific absorption.

With the proviso that 1 ng≈Δ0.1, R_(max) of the MIM is 0.014°, whichcorresponds to 4 ng/mm² of the CRP binding and 8% of a calculatedmaximum coverage of the SPR chip surface.

Effect of Free Phosphocholine

In order to study whether specific binding of a CRP to a molecularreceptor occurs, an effect of free phosphocholine (fPC) had beenresearched. For the molecularly imprinted monolayer, two sets of 300 μLof a binding buffer sample containing 200 nM of the CRP and 10 mM of thefPC were prepared. One sample was directly injected without undergoingpre-culture. The other sample was injected after undergoing pre-cultureat a room temperature for 15 minutes. FIG. 16 shows results for aneffect of fPC in CRP binding to the molecularly imprinted monolayer andthe molecularly non-imprinted monolayer. In the injection of the sample,the rapid decrease of the incidence angle was merely a result of aneffect of the fPC (data therefor are not illustrated) by deprivation ofcalcium ions from the surface in the form of a calcium-fPC ioniccompound. If there is no pre-culturing of the CRP and the fPC, theincidence angle shift (0.007°) was observed in the molecularly imprintedmonolayer. An amount of CRP binding corresponded to injection of 8.2 nMof the CRP according to a standard curve (FIG. 14, y=0.0016ln(x)+0.0368) for a binding ability of the molecularly imprintedmonolayer, and was almost half of R_(max) (0.014) of the molecularlyimprinted monolayer. In the case where the sample has been pre-cultured,no binding was observed on the molecularly imprinted monolayer. Thismeans that the fPC formed a complex with the CRP during thepre-culturing of the sample. The fPC-CRP complex cannot be bound to themolecular receptor on the molecularly imprinted monolayer. However, ifthere is no pre-culturing of fPC-CRP, the CRP can be partially bound toa PC receptor site. This shows that CRP binding to the molecularlyimprinted monolayer is very specific to the molecular receptorsynthesized through molecularly imprinting and click chemistry. When aCRP-fPC solution that had been pre-cultured for 15 minutes was injectedinto the molecularly non-imprinted monolayer, the binding was observedwith 0.004° of angle shift. The result was partially due to that the CRPbinding to the molecularly non-imprinted monolayer according to therelation shown in FIG. 14 is non-specific binding. This is probablybecause when the binding site of the CRP to phosphorylcholine is blockedby the fPC in the sample, absorption onto the surface of the molecularlynon-imprinted monolayer occurs.

In the molecularly non-imprinted monolayer, considering results of theLangmuir isotherm research presented in FIG. 15, which is not applied tothe Langmuir isotherm model, it is believed that binding of the CRP tothe molecularly non-imprinted monolayer involved not only thephosphorylcholine receptor-adjusted specific binding but alsonon-specific binding which is not bound via the phosphorylcholinereceptor.

Selectivity of the SPR Chip

Binding selectivity of a molecular receptor (molecularly imprintedstructure) synthesized in an illustrative embodiment has beenresearched. A 2% N-serum is injected. After the surface is stabilized,200 nM of a CRP is injected. In the binding of the CRP to the CM and themolecularly non-imprinted monolayer, the binding of the CRP was notoccurred after the 2% N-serum covers the surface. However, in themolecularly imprinted monolayer, incidence angle shift by 200 nM(0.020°) and 400 nM (0.032°) of the CRP was observed even after the 2%N-serum covers the surface (FIG. 17). This means that the molecularreceptor on the molecularly imprinted monolayer surface was notsubstituted with other serum proteins. In FIG. 14, aconcentration-dependent CRP binding curve shows that incidence angleshift is 0.013 in case of 200 nM of the CRP and 0.014 in case of 400 nMof the CRP. The difference in angle shift resulted from the followingdifferent measurement conditions: (1) the 2% N-serum improved stabilityof the binding state of the CRP to the molecular receptor throughprotein-protein interaction, and reduced removal of the bound CRP fromthe surface through washing; (2) instrumental deviation caused byinjection of a large amount of protein, which corresponds to ˜420 μg ofa serum protein in 300 μl of the 2% N-serum (based on 70 mg/ml oforiginal serum protein concentration). In order to identifyphosphorylcholine-specific binding of the CRP to the SPR chip under thepresence of an N-serum protein, 10 mM of the fPC was treated in thesample solution (2% N-serum+200 nM of the CRP) and pre-cultured for 15minutes. Prior to injection of the sample, 1% BSA was injected on thesurface for protection from non-specific binding by a large amount ofproteins in the serum. FIG. 18 shows that even if serum proteins aresimultaneously treated in the molecular receptor, the fPC completelyinhibited the CRP binding to the molecularly imprinted monolayer. Thismeans that the incidence angle shift by the CRP and the N-serum mixture,as observed in the molecularly imprinted monolayer, was caused from theCRP-receptor binding, and not non-specific binding of other proteins.When the 2% serum and 200 nM of the CRP were simultaneously treated,0.026° of angle shift was observed (FIG. 18). As a result, the CRP inserum proteins was specifically bound to the receptor and thus can beapplied to detection of CRPs in a human serum.

INDUSTRIAL APPLICABILITY

As described, the present disclosure relates to a method for preparing amolecularly imprinted structure, providing an improved effect indetection of pentraxin proteins such as a C-reactive protein, a serumamyloid P component, and PTX-3. The present disclosure may be applied tovarious types of molecularly imprinted polymers, using a complex of areactive group-pentraxin protein ligand capable of polymerizing apentraxin protein and a reactive ligand directly on a reactive surface.Also, it is possible to prepare a film and a bead for detection of apentraxin protein by using the method. Accordingly, it is possible tomore easily detect and purify a pentraxin protein by using a complex ofa molecularly imprinted reactive group-pentraxin protein ligand having astronger binding force than that of a ligand of a single pentraxinprotein.

What is claimed is:
 1. A chip for detection of a C-reactive protein, thechip comprising: a fixing layer formed on a metal substrate and havingan azide or an acetylene end group R¹; and a molecularly imprinted layerformed on the fixing layer and including R²—X-phosphocholine (wherein R²is an azide or an acetylene end group, and X is a spacer group having alength of about 0.1 nm to about 5 nm), wherein the phosphocholine groupincluded in the molecularly imprinted layer is arranged to correspond toa phosphocholine binding site of each molecule of the C-reactiveprotein, and wherein the fixing layer and the molecularly imprintedlayer may be bound through a click chemistry reaction between the endgroup R¹ of the fixing layer and R² included in R²—X-phosphocholine ofthe molecularly imprinted layer.
 2. The chip for detection of aC-reactive protein claimed in claim 1, wherein R² of the molecularlyimprinted layer is the acetylene group with the proviso that the endgroup R¹ of the fixing layer is the azide group, and R² of themolecularly imprinted layer is the azide group with the proviso that theend group R¹ of the fixing layer is the acetylene group.
 3. The chip fordetection of a C-reactive protein claimed in claim 1, wherein the fixinglayer is a polymer layer, a self assembled monolayer, or mica having theazide or the acetylene end group R¹ on the surface thereof.
 4. The chipfor detection of a C-reactive protein claimed in claim 3, wherein theself assembled monolayer may be formed including HS—Y—R¹ (wherein R¹ isthe azide or the acetylene end group, and Y is a spacer group having alength of about 0.1 nm to about 5 nm).
 5. The chip for detection of aC-reactive protein claimed in claim 1, wherein the molecularly imprintedlayer is formed including acetylene-C₁₋₃₀alkylene-phosphorylcholine,propargyloxy-C₁₋₃₀alkylene-phosphorylcholine, orazido-C₁₋₃₀alkylene-phosphorylcholine.
 6. The chip for detection of aC-reactive protein claimed in claim 4, wherein the self assembledmonolayer is formed including azido-C₁₋₃₀alkylene-1-thiol,acetylene-C₁₋₃₀alkylene-1-thiol, or propargyloxy-C₁₋₃₀alkylene-1-thiol.7. A method for preparing a chip for detection of a C-reactive protein,the method comprising: forming a fixing layer having an azide or anacetylene end group R¹ on a metal substrate; mixing a C-reactive proteinand a molecularly imprinted material containing R²—X-phosphocholine(wherein R² is an azide or an acetylene end group, and X is a spacergroup having a length of about 0.1 nm to 5 nm) to obtain a mixturesolution of the molecularly imprinted material/the C-reactive protein;immersing the metal substrate, on which the fixing layer is formed, inthe mixture solution of the molecularly imprinted material/theC-reactive protein to obtain a complex layer of the metal substrate/thefixing layer/the molecularly imprinted material/the C-reactive proteinthrough a click chemistry reaction between the end group R¹ of thefixing layer and R² included in R²—X-phosphocholine of the molecularlyimprinted material; and removing the C-reactive protein from the complexlayer to obtain a molecularly imprinted layer.